Control of transport by epithelia

我们研究动物用来调节跨上皮细胞的离子和水运输的机制。我们使用的主要模型系统是一种耐寒的硬骨鱼，它经常出现在北美的河口，对极端环境(盐度、温度、低氧、污染)具有很强的抵抗力，而粘鼠是一种重要的生理和基因组模式物种，也是重要的生物指示物种。我们使用的渗透压调节功能的模型有富含盐转运离子细胞的扁平上皮、成人的穹隆上皮和胚胎的卵黄囊上皮。这项研究的重点是揭示直接或通过激酶级联来调节运输蛋白的调节蛋白(激酶和磷酸酶)。目的1.我们将发现控制阴离子通道囊性纤维化跨膜电导调节蛋白(CFTR)的转运蛋白复合体的调控成分，以及包括多种激酶和钙依赖抑制通路的Na，K，2Cl共转运体(NKCC1)和氯化钠共转运体(NCC)。这些途径被渗透刺激、神经递质和激素激活。最重要的假设是，膜运输蛋白在明显多余的调节离子运输的途径中有多个调节成分，但在像Fundulus和不那么耐寒的热带鱼(尼罗罗非鱼、尼罗罗非鱼)等耐寒动物中，环境的极端变化决定了哪些途径最适合在极端环境中运行。目的2.我们将利用简单的盐度变化和转运蛋白的免疫细胞化学，将上述高度调控的运输系统的细胞动力学覆盖在上面，以产生细胞继承与现有离子细胞的重塑以及它们在淡水、海水和高盐条件下的各种表型变化。目的3.我们将利用Fundulus胚胎(也是耐盐性强且富含离子细胞)的卵黄囊膜，通过注射短干扰mRNA(SiRNA)来检测调控途径，以阻断卵黄囊中特定的激酶/磷酸酶基因(经定量PCR证实)，并观察运输调节(低渗、高渗休克、肾上腺素能激动剂)是如何选择性地被破坏以确定调控途径的关键组成部分。重要性：了解离子传输及其调节也为渔业和水产养殖提供信息，促进比较生理学的发展。这项研究将揭示调控离子转运蛋白的调节蛋白之间的新关系，特别是CFTR(与囊性纤维化有关)、NKCC(与假性醛固酮减少症有关)和NCC(与Gitelman综合征有关)。这项工作将为加拿大的研究贡献19个HQP(理学学士和理学硕士)。

We study mechanisms that animals use to regulate ion and water transport across epithelia. The main model system we use is a hardy teleost fish that frequents estuaries of N. America and is robust to environmental extremes (salinity, temperature, low oxygen, pollution), the mummichog (Fundulus heteroclitus), an important physiological and genomic model species and important as a bioindicator species. The models of the gill osmoregulatory function we use are flat epithelia rich in salt transporting ionocytes, the opercular epithelium in adults and the yolk sac epithelium of embryos. The research focuses on revealing regulatory proteins (kinases and phosphatases) that modulate the transport proteins directly or via kinase cascades. Aim 1. We will discover the regulatory components of the transport complexes that control the anion channel Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the cotransporters Na,K,2Cl cotransporter (NKCC1) and NaCl cotransporter (NCC) which include many kinases as well as a calcium-dependent inhibitory pathway. These pathways are activated by osmotic stimuli, neurotransmitters and hormones. The overarching hypothesis is that membrane transport proteins have multiple regulatory components in apparently redundant pathways that regulate ion transport, but in hardy animals such as Fundulus and less hardy tropical fish (Nile tilapia,Oreochromis nilotica), the extreme variation in the environment determines which pathways are best-suited for operation in extreme environments. Aim 2. We will overlay the above with cellular dynamics of this highly regulated transport system using simple salinity changes and immunocytochemistry of transporter proteins to generate cellular succession vs. remodeling of existing ionocytes and their various phenotypic variations in freshwater, seawater and hypersaline conditions. Aim 3. We will use the yolk sac membrane of Fundulus embryos (also salt hardy and rich in ionocytes) to test regulatory pathways using short interfering mRNA (siRNA) injection to block specific kinase/phosphatase genes (confirmed by quantitative PCR) in the yolk sac and see how transport regulation (hypotonic, hypertonic shock, adrenergic agonists) are selectively disrupted to identify the critical components of the regulatory pathways. Importance: Understanding of ion transport and its regulation also informs fisheries and aquaculture and advances comparative physiology. The research will reveal new relationships among regulatory kinases controlling ion transport proteins also associated with disease, particularly CFTR (responsible for cystic fibrosis), NKCC (implicated in pseudohypoaldosteronism) and NCC (involved in Gitelman's syndrome). The work will contribute 19 HQP (BSc and MSc) to Canadian research.******